Membrane fusion is a fundamental biological process for organelle formation, nutrient uptake, and the secretion of hormones and neurotransmitters. Imbalances in these processes give rise to important diseases, such as diabetes, obesity, and mood disorders. A major advance has been made in the first three years of this project with the discovery that in exocytosis, Complexin (CPX) and Synaptotagmin1 (SYT1) couple calcium ion to otherwise spontaneous fusion by the ubiquitous SNARE fusion machinery. By flipping exocytic SNAREs to the outside of cells, and measuring their activity in fusing whole cells, we create a compositionally virgin environment in which factors such as CPX and SYT1 can be added to the medium or expressed from the cells, one at a time or in combination, to reveal their roles with clarity. For the current grant period (commencing 7/1/04), we proposed to survey a broad range of known in vivo regulators with unknown molecular mechanism using the flipped SNARE assay. As a result, we are now narrowing the focus to capitalize on our discovery of a minimal machine for calcium-triggered exocytosis over the next five years. Specifically, we will 1) define the structural requirements for minimal calcium regulation of SNARE-dependent fusion by Complexin and Synaptotagmin using the flipped SNARE assay, and test the in vivo relevance of these findings;2) define the molecular mechanism of the Complexin/Synaptotagmin clamp using the Surface Forces Apparatus (SFA);and comprehensively and in high throughput explore the functional biology of the broad Synaptotagmin and Complexin families by establishing which members have the capacity to clamp control fusion by which SNARE proteins. PUBLIC HEALTH RELEVANCE: Membrane fusion is a fundamental biological process for organelle formation, nutrient uptake, and the secretion of hormones and neurotransmitters. It is central to vesicular transport, storage, and release in many areas of endocrine and exocrine physiology, and imbalances in these processes give rise to important diseases, such as diabetes. Membrane fusion is the result of a highly orchestrated series of protein-protein interactions [5] which work together to activate and regulate the membrane fusion machinery known as SNARE proteins. Transport vesicles dock closely and firmly - within molecular contact distance of the target bilayer - as the cognate SNAREs zip-up to form a four helix bundle between the two membranes (termed a trans-SNARE complex or SNAREpin). When the SNARE complex fully zips up the bilayers are merged and the SNARE complex now emanates from the single, combined membrane. In regulated exocytosis, there are additional proteins added to this "core" machinery that appear to freeze the process at intermediate stages to allow rapid mobilization from these intermediates (for example, upon calcium entry) to allow a prompt bolus of secretion (for example, of insulin). How such proteins accomplish this, or potentially contribute directly to the bilayer fusion event itself, has been difficult to discern with any precision. The long-term goal of this project is to bridge the gap, utilizing functional studies to validate whether and how regulatory proteins influence exocytosis through their interactions with SNARE proteins.